Recursive Directional Ligation by Plasmid Reconstruction Allows Rapid and Seamless Cloning of Oligomeric Genes

• Published in: Biomacromolecules Vol. 11; no. 4; pp. 944 - 952
• Main Authors: McDaniel, Jonathan R, MacKay, J. Andrew, Quiroz, Felipe García, Chilkoti, Ashutosh
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 12.04.2010
• Subjects: Biological and medical sciences; Biotechnology; Cloning, Molecular; DNA Restriction Enzymes - metabolism; Elastin - chemistry; Elastin - genetics; Elastin - metabolism; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Genes - genetics; Genetic engineering; Genetic technics; Humans; Methods. Procedures. Technologies; Peptides - metabolism; Phase Transition; Plasmids - genetics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Plasmid; Gene; Molecular cloning; Oligomer; Repeated sequence
• ISSN: 1525-7797; 1526-4602
• DOI: 10.1021/bm901387t

This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.